Field of the Invention
The present application relates to methods for purifying recombinant coagulation factor proteins, including for example, prothrombin (Factor II). In embodiments, the methods provide purified prothrombin that exhibit increased bioactivity and reduced levels of thrombin, thereby increasing the safety of the prothrombin. Also provided are purified recombinant coagulation factor proteins.
Background of the Invention
Coagulation factors for therapeutic use (e.g., for the treatment of hemophilia and other blood disorders) can be obtained from human plasma, though purification can be complex. Even with extensive safety measures and testing of blood-derived protein products, contamination with infectious viruses or prions cannot be ruled out. Therefore, is highly desirable to produce human therapeutic proteins from recombinant cells grown in media without animal derived components. This is not always straightforward as many proteins require a mammalian host to be produced in a fully functional form, i.e. to be correctly post-translationally modified.
Factor II (prothrombin) is a coagulation factor necessary for blood clotting. In the extrinsic (or tissue factor) blood coagulation pathway, tissue factor triggers a coagulation cascade in which prothrombin, in the presence of Factor Xa, FactorVa, phospholipids, and Ca2+ ions, becomes the activated form, Factor IIa (thrombin). Thrombin acts through multiple pathways to stop bleeding by converting fibrinogen to fibrin clots, and participating in other pro-coagulation pathways.
During the production and purification of recombinant prothrombin protein, thrombin can be present as a product-related, impurity. Levels of free thrombin generally must be kept at low levels to increase product safety. In addition, recombinant prothrombin contains 10 gamma-carboxyglutamic acid “Gla-residues” in the N-terminal domain. These residues are thought to chelate Ca2+ ions, which mediates the protein's binding to phospholipid membranes, a prerequisite for prothrombin activation to thrombin.
The inventors of the present application have identified a need for purification methods that provide recombinant purified prothrombin having high levels of γ-carboxylated glutamic acid residues and low thrombin contamination.